Gene involved in pyrimidine biosynthesis in plants

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding an OMP decarboxylase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the OMP decarboxylase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the OMP decarboxylase in a transformed host cell.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/675018, filed Sep. 28, 2000, hereby incorporated byreference herein in its entirety, which claims the benefit of U.S.Provisional Application No. 60/156901, filed Sep. 30,1999.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid molecules codingfor an enzyme involved in pyrimidine biosynthesis in plants, especiallyin seeds.

BACKGROUND OF THE INVENTION

[0003] Orotidine 5′-monophosphate decarboxylase (OMP decarboxylase)catalyzes the final reaction in pyrimidine nucleotide biosynthesis,converting OMP to uridine 5′-monophosphate (UMP). In eukaryotes, thisenzyme also performs the next-to-last step of linkingphosphoribosyl-pyrophosphate (PRPP) to orotate to form OMP (Reyes andGuganig (1975) J Biol Chem 250:5097-108; Traut et al. (1980)Biochemistry 19:6062-8). The enzyme is a target for feedback inhibitionwherein UTP and UMP both reduce its activity. In prokaryotes, incontrast, there is no feedback inhibition, and the last two enzymaticreactions are not coupled.

[0004] Nucleotides are required for the synthesis of DNA and RNA, andare indirectly responsible for protein synthesis, due to the requirementfor ribosomes and tRNAs in translation. Therefore, pyrimidinebiosynthesis is a key metabolic pathway in all eukaryotes. Manipulationof this pathway is nevertheless possible since mutations to key enzymescan be partially overcome by feeding cells having potentially lethalmutations with CTP, UTP, or their mono- or diphosphate derivatives.Inhibitors of OMP decarboxylase have been identified which vary in theirefficacy among different organisms, implying that engineering of theactive site may yield enzymes that are more or less sensitive toinhibition (Shostak and Jones (1992) Biochemistry 31:12155-61). It isbelieved that overexpression or inhibition of OMP decarboxylase inplants may be useful for enhancing growth rates, developing newherbicides, developing new fungicides, developing new insecticides, orselectively altering development of individual organs.

SUMMARY OF THE INVENTION

[0005] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a first nucleotide sequence encoding a polypeptide of at least 200amino acids having at least 85% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, or (b) a secondnucleotide sequence comprising the complement of the first nucleotidesequence.

[0006] In a second embodiment, it is preferred that the isolatedpolynucleotide of the claimed invention comprises a first nucleotidesequence which comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11 that codes for thepolypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, and 12.

[0007] In a third embodiment, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence of at least one of 800(preferably at least one of 500, most preferably at least one of 400)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11 and thecomplement of such nucleotide sequences.

[0008] In a fourth embodiment, this invention relates to a chimeric genecomprising an isolated polynucleotide of the present invention operablylinked to at least one suitable regulatory sequence.

[0009] In a fifth embodiment, the present invention concerns an isolatedhost cell comprising a chimeric gene of the present invention or anisolated polynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

[0010] In a sixth embodiment, the invention also relates to a processfor producing an isolated host cell comprising a chimeric gene of thepresent invention or an isolated polynucleotide of the presentinvention, the process comprising either transforming or transfecting anisolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the present invention.

[0011] In a seventh embodiment, the invention concerns an OMPdecarboxylase polypeptide of at least 200 amino acids comprising atleast 85% identity based on the Clustal method of alignment compared toa polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6,8, 10, and 12.

[0012] In an eighth embodiment, the invention relates to a method ofselecting an isolated polynucleotide that affects the level ofexpression of an OMP decarboxylase polypeptide or enzyme activity in ahost cell, preferably a plant cell, the method comprising the steps of:(a) constructing an isolated polynucleotide of the present invention oran isolated chimeric gene of the present invention; (b) introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;(c) measuring the level of the OMP decarboxylase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; and(d) comparing the level of the OMP decarboxylase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide withthe level of the OMP decarboxylase polypeptide or enzyme activity in thehost cell that does not contain the isolated polynucleotide.

[0013] In a ninth embodiment, the invention concerns a method ofobtaining a nucleic acid fragment encoding a substantial portion of anOMP decarboxylase polypeptide, preferably a plant OMP decarboxylasepolypeptide, comprising the steps of: synthesizing an oligonucleotideprimer comprising a nucleotide sequence of at least one of 800(preferably at least one of 500, most preferably at least one of 400)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a substantial portion of an OMP decarboxylase amino acidsequence.

[0014] In a tenth embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding an OMP decarboxylase polypeptidecomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

[0015] In an eleventh embodiment, this invention concerns a composition,such as a hybridization mixture, comprising an isolated polynucleotideof the present invention.

[0016] In a twelfth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the chimeric gene of the present invention or anexpression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the OMP decarboxylasepolynucleotide in an amount sufficient to complement a null mutant toprovide a positive selection means.

[0017] In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of an OMP decarboxylase in a host cellcomprising: (a) transforming a host cell with a chimeric gene of thepresent invention; and (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof the OMP decarboxylase in the transformed host cell.

[0018] A further embodiment of the instant invention is a method forevaluating at least one compound for its ability to inhibit the activityof an enzyme involved in primidine biosynthesis, the method comprisingthe steps of: (a) transforming a host cell with a chimeric genecomprising a nucleic acid fragment encoding an OMP decarboxylasepolypeptide, operably linked to suitable regulatory sequences; (b)growing the transformed host cell under conditions that are suitable forexpression of the chimeric gene wherein expression of the chimeric generesults in production of an OMP decarboxylase in the transformed hostcell; (c) optionally purifying the OMP decarboxylase polypeptideexpressed by the transformed host cell; (d) treating the OMPdecarboxylase polypeptide with a compound to be tested; and (e)comparing the activity of the OMP decarboxylase polypeptide that hasbeen treated with a test compound to the activity of an untreated OMPdecarboxylase polypeptide, thereby selecting compounds with potentialfor inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0019] The invention can be more fully understood from the followingdetailed description and the accompanying drawings and Sequence Listingwhich form a part of this application.

[0020]FIG. 1 shows a comparison of the amino acid sequences set forth inSEQ ID NOs: 6, 8, 10, and 12, and the Arabidopsis thaliana and Nicotianatabacum (NCBI General Identifier No. gi 2499945 and gi 2499946,respectively) sequences (SEQ ID NOs: 13 and 14, respectively).

[0021] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Orotidine-5-PhosphateDecarboxylase SEQ ID NO: Protein Clone Designation (Nucleotide) (AminoAcid) Corn OMP Decarboxylase p0127.cntbe57r 1 2 Rice OMP Decarboxylasersl1n.pk004.j19 3 4 Soybean OMP Decarboxylase sf11.pk135.i17 5 6 WheatOMP Decarboxylase wl1n.pk0029.c7 7 8 Corn OMP Decarboxylasep0127.cntbe57r:fis 9 10 Rice OMP Decarboxylase rsl1n.pk004.j19:fis 11 12

[0022] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 800contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs: 1, 3, 5, 7, 9, and 11, or the complement ofsuch sequences.

[0024] The term “isolated” polynucleotide refers to a polynucleotidethat is substantially free from other nucleic acid sequences, such asother chromosomal and extrachromosomal DNA and RNA, that normallyaccompany or interact with it as found in its naturally occurringenvironment. Isolated polynucleotides may be purified from a host cellin which they naturally occur. Conventional nucleic acid purificationmethods known to skilled artisans may be used to obtain isolatedpolynucleotides. The term also embraces recombinant polynucleotides andchemically synthesized polynucleotides.

[0025] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques.

[0026] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0027] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0028] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 800 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

[0029] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 800 (preferably at least one of500, most preferably at least one of 400) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 9, and 11, and the complement of such nucleotidesequences may be used in methods of selecting an isolated polynucleotidethat affects the expression of an OMP decarboxylase polypeptide in ahost cell. A method of selecting an isolated polynucleotide that affectsthe level of expression of a polypeptide in a virus or in a host cell(eukaryotic, such as plant or yeast, prokaryotic such as bacterial) maycomprise the steps of: constructing an isolated polynucleotide of thepresent invention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide with the level of a polypeptideor enzyme activity in a host cell that does not contain the isolatedpolynucleotide.

[0030] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0031] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least about70% identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences disclosed herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0032] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0033] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0034] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0035] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0036] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0037] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0038] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0039] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0040] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0041] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0042] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0043] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0044] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0045] “Null mutant” refers here to a host cell which either lacks theexpression of a certain. polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

[0046] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0047] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0048] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0049] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0050] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0051] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) first nucleotide sequence encoding a polypeptide of at least 200amino acids having at least 85% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, or (b) a secondnucleotide sequence comprising the complement of the first nucleotidesequence.

[0052] Preferably, the first nucleotide sequence comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9, and 11, that codes for the polypeptide selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

[0053] Nucleic acid fragments encoding at least a portion of several OMPdecarboxylase have been isolated and identified by comparison of randomplant cDNA sequences to public databases containing nucleotide andprotein sequences using the BLAST algorithms well known to those skilledin the art. The nucleic acid fragments of the instant invention may beused to isolate cDNAs and genes encoding homologous proteins from thesame or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

[0054] For example, genes encoding other OMP decarboxylase, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

[0055] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least one of 800 (preferably oneof at least 500, most preferably one of at least 400) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11 and the complement ofsuch nucleotide sequences may be used in such methods to obtain anucleic acid fragment encoding a substantial portion of an amino acidsequence of a polypeptide.

[0056] The present invention relates to a method of obtaining a nucleicacid fragment encoding a substantial portion of an OMP decarboxylasepolypeptide, preferably a substantial portion of a plant OMPdecarboxylase polypeptide, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 800 (preferably at least one of 500, most preferably at least one of400) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a portion of an OMP decarboxylase polypeptide.

[0057] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0058] In another embodiment, this invention concerns viruses and hostcells comprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

[0059] As was noted above, the nucleic acid fragments of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of pyrimidinebiosynthesis or metabolism in those cells.

[0060] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

[0061] Plasmid vectors comprising the instant isolated polynucleotide(or chimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0062] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0063] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0064] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0065] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

[0066] In another embodiment, the present invention concerns apolypeptide of at least 200 amino acids that has at least 85% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, and12.

[0067] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded OMP decarboxylase. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 6).

[0068] Additionally, the instant polypeptides can be used as a target tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze a key step in pyrimidinebiosynthesis. Accordingly, inhibition of the activity of one or more ofthe enzymes described herein could lead to inhibition of plant growth.Thus, the instant polypeptides could be appropriate for new herbicidediscovery and design.

[0069] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0070] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0071] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0072] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeRes. 5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

[0073] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0074] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0075] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0076] The disclosure of each reference set forth herein is incorporatedherein by reference in its entirety.

Example 1

[0077] Composition of cDNA Libraries; Isolation and Sequencing of cDNAClones

[0078] cDNA libraries representing mRNAs from various corn, rice,soybean, and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean, and Wheat Library Tissue Clone p0127 Nucellus tissue, 5 daysafter silking, p0127.cntbe57r screened 1 rsl1n Rice (Oryza sativa, YM)15 day old rsl1n.pk004.j19 seedling normalized* sf11 Soybean ImmatureFlower sfl1.pk135.i17 wl1n Wheat Leaf From 7 Day Old Seedling*wl1n.pk0029.c7 p0127 Nucellus tissue, 5 days after p0127.cntbe57r:fissilking, screened 1 rsl1n Rice (Oryza sativa, YM) 15 day oldrsl1n.pk004.j19:fis seedling normalized sf11 Soybean Immature Flowersfl1.pk135.i17:fis

[0079] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0080] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0081] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Ty1 transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0082] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phrep/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

Example 2

[0083] Identification of cDNA Clones

[0084] cDNA clones encoding OMP decarboxylase were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

[0085] ESTs submitted for analysis are compared to the genbank databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTn algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402.) against the DuPont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the tBLASTn algorithm. ThetBLASTn algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

Example 3

[0086] Characterization of cDNA Clones Encoding OMP Decarboxylase

[0087] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs toOMP decarboxylase from Arabidopsis thaliana and Nicotiana tabacum (NCBIGeneral Identifier No. gi 2499945 and gi 2499946, respectively). Shownin Table 3 are the BLAST results for individual ESTs (“EST”), thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), the sequences of contigs assembled from two or more ESTs(“Contig”), sequences of contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding an entire protein derived froman FIS, a contig, or an FIS and PCR (“CGS”): TABLE 3 BLAST Results forSequences Encoding Polypeptides Homologous to OMP Decarboxylase BLASTpLog Score Clone Status gi2599945 gi2499946 p0127.cntbe57r EST 25.70rsl1n.pk004.j19 EST 37.70 sfl1.pk135.i17 EST >180.00 wl1n:pk0029.c7 FIS167.00

[0088] The sequence of the entire cDNA insert in the clones listed inTable 3 was determined. Further sequencing and searching of the DuPontproprietary database allowed the identification of other corn, rice,soybean and/or wheat clones encoding OMP decarboxylase. The BLASTXsearch using the EST sequences from clones listed in Table 4 revealedsimilarity of the polypeptides encoded by the cDNAs to OMP decarboxylasefrom Arabidopsis thaliana (NCBI General Identifier No. gi 2599945).Shown in Table 4 are the BLAST results for individual ESTs (“EST”), thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), sequences of contigs assembled from two or more ESTs(“Contig”), sequences of contigs assembled from an FIS and one or moreESTs (“Contig*”), or sequences encoding the entire protein derived froman FIS, a contig, or an FIS and PCR (“CGS”): TABLE 4 BLAST Results forSequences Encoding Polypeptides Homologous to OMP Decarboxylase BLASTpLog Score Clone Status gi2599945 p0127.cntbe57r:fis FIS >180.00rsl1n.pk004.j19:fis CGS 172.00

[0089]FIG. 1 presents an alignment of the amino acid sequences set forthin SEQ ID NOs: 6, 8, 10, and 12, and the Arabidopsis thaliana andNicotiana tabacum (NCBI General Identifier No. gi 2499945 and gi2499946, respectively) sequences (SEQ ID NOs: 13 and 14, respectively).The data in Table 5 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, and12, and the Arabidopsis thaliana and Nicotiana tabacum (NCBI GeneralIdentifier No. gi 2499945 and gi 2499946, respectively) sequences (SEQID NOs: 13 and 14, respectively). TABLE 5 Percent Identity of Amino AcidSequences Deduced From the Nucleotide Sequences of cDNA Clones EncodingPolypeptides Homologous to OMP Decarboxylase Percent Identity to SEQ IDNO. gi2599945 gi2599946 2 52.6% 4 79.5% 6 75.6% 8 69.2% 10 67.0% 1265.7%

[0090] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a OMP decarboxylase.These sequences represent the first corn, soybean, and wheat sequencesencoding OMP decarboxylase known to Applicant.

Example 4

[0091] Expression of Chimeric Genes in Monocot Cells

[0092] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue□; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase□ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0093] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0094] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0095] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 □m in diameter) are coatedwith DNA using the following technique. Ten □g of plasmid DNAs are addedto 50 □L of a suspension of gold particles (60 mg per mL). Calciumchloride (50 □L of a 2.5 M solution) and spermidine free base (20 □L ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 □L of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 □L of ethanol. Analiquot (5 □L) of the DNA-coated gold particles can be placed in thecenter of a Kapton□ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic□ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0096] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0097] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0098] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5

[0099] Expression of Chimeric Genes in Dicot Cells

[0100] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the □ subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0101] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0102] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0103] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0104] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic□PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0105] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0106] To 50 □L of a 60 mg/mL 1 □m gold particle suspension is added (inorder): 5 □L DNA (1 □g/□L), 20 □L spermidine (0.1 M), and 50 □L CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 □L 70% ethanol andresuspended in 40 □L of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five □L of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0107] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0108] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6

[0109] Expression of Chimeric Genes in Microbial Cells

[0110] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0111] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 □g/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase□ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 □L of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 □g/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0112] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-□-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 □L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One □g ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 7

[0113] Evaluating Compounds for Their Ability to Inhibit the Activity ofOMP Decarboxylase

[0114] The polypeptides described herein may be produced using anynumber of methods known to those skilled in the art. Such methodsinclude, but are not limited to, expression in bacteria as described inExample 6, or expression in eukaryotic cell culture, in planta, andusing viral expression systems in suitably infected organisms or celllines. The instant polypeptides may be expressed either as mature formsof the proteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

[0115] Purification of the instant polypeptides, if desired, may utilizeany number of separation technologies familiar to those skilled in theart of protein purification. Examples of such methods include, but arenot limited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed, enzyme or an affinityresin containing ligands which are specific for the enzyme. For example,the instant polypeptides may be expressed as a fusion protein coupled tothe C-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents include□-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond□ affinityresin or other resin.

[0116] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for OMP decarboxylase are presented byStrych et al. (1994) Curr Microbiol 29:353-9; Seymour et al. (1994)Biochemistry 33:5268-74; Shostak and Jones (1992) Biochemistry31:12155-61; and Traut et al. (1980) Biochemistry 19:6062-8.

1 14 1 510 DNA Zea mays unsure (9) n = A, C, G or T 1 gtacaaagncacttcctgct ctcgctccgc cgccgccgcc tccctcccca gtcgatcacc 60 aaacctcagtccaaactcca aacccccgcc gcatcagaaa aaaaccctag gccatggacg 120 ccgcggcgctggagtcgctc atcctggacc tccacgccat cgaggtcgtg aagctgggct 180 ccttcacgctcaagtccggc atcaaatcgc ccatctacct cgacctccgc gcgctcgnct 240 cccacccgcgcctgctctcc gncgtcgcct cgctccttca cgcgctcccg gccacgcgcc 300 cctacggcctcgtctgcggt gncccctaca ccgcgctccc catcgccgnc gncctctccg 360 tcgaccgctcaatccccatg ctcatgcgcc gcaaggaggt caaggnccac ggaccgcaag 420 tccatcgagggctcttcagc cggggacacc gnctatatng agactcgcac agtggnnctc 480 ggctcngacgcgccgtcggc gagggctgcg 510 2 97 PRT Zea mays UNSURE (39) Xaa = ANY AMINOACID 2 Ala Ala Leu Glu Ser Leu Ile Leu Asp Leu His Ala Ile Glu Val Val 15 10 15 Lys Leu Gly Ser Phe Thr Leu Lys Ser Gly Ile Lys Ser Pro Ile Tyr20 25 30 Leu Asp Leu Arg Ala Leu Xaa Ser His Pro Arg Leu Leu Ser Xaa Val35 40 45 Ala Ser Leu Leu His Ala Leu Pro Ala Thr Arg Pro Tyr Gly Leu Val50 55 60 Cys Gly Xaa Pro Tyr Thr Ala Leu Pro Ile Ala Xaa Xaa Leu Ser Val65 70 75 80 Asp Arg Ser Ile Pro Met Leu Met Arg Arg Lys Glu Val Lys XaaHis 85 90 95 Gly 97 3 514 DNA Oryza sativa unsure (376) n = A, C, G or T3 ctgcttttgc ttgctgaaat gagctcggct ggcaaccttg ctcatggaga gtacactgct 60gcagctgtaa agattgctga gcaacattct gattttgtaa ttggatttat atccgttaat 120ccagcatctt ggtcagttgc gccatcaagt ccagcattta tccatgccac tcctggagtg 180cagatggttt ctggaggaga tgctcttggt caacagtaca atacccctca ttctgttata 240aacgacaaga ggcaagtgac ataattatag tccggacgag ggattataaa ggcgaagtaa 300tccagcccga gaccgcgagg gaagtaccgc atccaagggt gggggagcaa aacaatccag 360ctttgccatg agaaantgag aatngtgttt aggcaatggt tggttcnagc ttatgattta 420ttataaccaa gaataattaa gccangattg cnnataaagc cgggattaat antnaagctg 480ccatanaaat aaactgtgna gttggttgnt ttgg 514 4 83 PRT Oryza sativa 4 LeuLeu Leu Leu Ala Glu Met Ser Ser Ala Gly Asn Leu Ala His Gly 1 5 10 15Glu Tyr Thr Ala Ala Ala Val Lys Ile Ala Glu Gln His Ser Asp Phe 20 25 30Val Ile Gly Phe Ile Ser Val Asn Pro Ala Ser Trp Ser Val Ala Pro 35 40 45Ser Ser Pro Ala Phe Ile His Ala Thr Pro Gly Val Gln Met Val Ser 50 55 60Gly Gly Asp Ala Leu Gly Gln Gln Tyr Asn Thr Pro His Ser Val Ile 65 70 7580 Asn Asp Lys 83 5 1730 DNA Glycine max 5 gcacgaggtt ttcctttactggtaagttgt aaccctataa gccgttgctc ccaccgccgc 60 cgcgcagatc tccgtttcaacttgggttat ctctaaggtc ctaaacaatc ctctttcaaa 120 aacataccga gaaaagtgtggaaatgacga caccatcatt ggtagagtct ctagttcttc 180 aactccatga gatctcagctgtcaaatttg gcaacttcaa gctcaaatct ggcatcttct 240 caccaatcta catagacctccgcctcatca tatcttaccc ttctctcctc caacagatct 300 ctcaaaccct tatttcttcagtctcttcca cttcctttga cctcgtatgc ggtgtccctt 360 acactgcctt acccattgctacatgtgtct ctcttgctca gaacattccc atggtcatgc 420 gccgcaaaga aatcaaagattatggcactg ctaaagctat tgaaggcgat ttcaagcctg 480 gccaaagttg cttaatcattgaggatttgg ttaccagtgg cacgtcagtt ttggaaactg 540 cggcgccatt gcgtgctgtgggattaaaga tcagtgatgc tgttgtgttg atcgatagag 600 agcaaggtgg cagagaaaacttggaggaga atggcatcaa gctgcatgca attattaaat 660 tgactgaaat ggtgaaaattttgggcaatc acgggaagct tgatgaagag atggtagggg 720 ttgttacgaa gttcttagaggataatcgta aggttgctgc tttggcaaag gtggagaagc 780 ctgtaactaa ggtcaaagctttgccatttg gggagagggc taagctgtcg aagaatccaa 840 tgggaaagag gttgtttgagataatggctg agaaggagag taatctatgt ttggctgctg 900 atgttggaac tgcagctgaattgcttgaaa ttgctgagaa ggttggacct gagatatgct 960 tgctgaagac tcatgtggatatttttccag attttactgc tgattttggc tctaagcttc 1020 tctcgattgc agaaaaacataacttcttaa tctttgagga tcgtaaattt gctgatattg 1080 gcaacacagt gaccatgcaatatgaaggag gggtttttcg tatattggat tgggctcata 1140 tagtaaatgc tcacataatctcaggtcctg gaattgttga tggattaaaa ttgaagggtt 1200 tacctcgtgg taggggtctattactgcttg ctgaaatgag ctcagctggt aaccttgcca 1260 agggagatta tacaacttctgcagtaaaaa ttgctgagga tcattctgac tttgtaattg 1320 gcttcatctc agtcaatcctgcatcatggc caggggcacc aataaatcct tctttcattc 1380 aagcaacccc tggagttcaaatggtaactg gtggcgatgc tttagggcag caatataaca 1440 ctccatattc tgtgatccatgataggggca gtgacatcat catcgtggga cgtggcatca 1500 tcaaagcagc aaaccatgctgagatagctc gtgaatatcg tcttcaagga tggaatgcat 1560 atttggctaa atgtaattgatgcctgcatt cctagaataa aattatgagc ttaaattatg 1620 ttttaatggg acatctgatctcactgtaac ccagatgaat aaggtcttgg ggtacaatat 1680 gaagacattt ttcggttggaatattgaaaa aaaaaaaaaa aaaaaaaaaa 1730 6 478 PRT Glycine max 6 Met ThrThr Pro Ser Leu Val Glu Ser Leu Val Leu Gln Leu His Glu 1 5 10 15 IleSer Ala Val Lys Phe Gly Asn Phe Lys Leu Lys Ser Gly Ile Phe 20 25 30 SerPro Ile Tyr Ile Asp Leu Arg Leu Ile Ile Ser Tyr Pro Ser Leu 35 40 45 LeuGln Gln Ile Ser Gln Thr Leu Ile Ser Ser Val Ser Ser Thr Ser 50 55 60 PheAsp Leu Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Thr 65 70 75 80Cys Val Ser Leu Ala Gln Asn Ile Pro Met Val Met Arg Arg Lys Glu 85 90 95Ile Lys Asp Tyr Gly Thr Ala Lys Ala Ile Glu Gly Asp Phe Lys Pro 100 105110 Gly Gln Ser Cys Leu Ile Ile Glu Asp Leu Val Thr Ser Gly Thr Ser 115120 125 Val Leu Glu Thr Ala Ala Pro Leu Arg Ala Val Gly Leu Lys Ile Ser130 135 140 Asp Ala Val Val Leu Ile Asp Arg Glu Gln Gly Gly Arg Glu AsnLeu 145 150 155 160 Glu Glu Asn Gly Ile Lys Leu His Ala Ile Ile Lys LeuThr Glu Met 165 170 175 Val Lys Ile Leu Gly Asn His Gly Lys Leu Asp GluGlu Met Val Gly 180 185 190 Val Val Thr Lys Phe Leu Glu Asp Asn Arg LysVal Ala Ala Leu Ala 195 200 205 Lys Val Glu Lys Pro Val Thr Lys Val LysAla Leu Pro Phe Gly Glu 210 215 220 Arg Ala Lys Leu Ser Lys Asn Pro MetGly Lys Arg Leu Phe Glu Ile 225 230 235 240 Met Ala Glu Lys Glu Ser AsnLeu Cys Leu Ala Ala Asp Val Gly Thr 245 250 255 Ala Ala Glu Leu Leu GluIle Ala Glu Lys Val Gly Pro Glu Ile Cys 260 265 270 Leu Leu Lys Thr HisVal Asp Ile Phe Pro Asp Phe Thr Ala Asp Phe 275 280 285 Gly Ser Lys LeuLeu Ser Ile Ala Glu Lys His Asn Phe Leu Ile Phe 290 295 300 Glu Asp ArgLys Phe Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr 305 310 315 320 GluGly Gly Val Phe Arg Ile Leu Asp Trp Ala His Ile Val Asn Ala 325 330 335His Ile Ile Ser Gly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly 340 345350 Leu Pro Arg Gly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser Ser Ala 355360 365 Gly Asn Leu Ala Lys Gly Asp Tyr Thr Thr Ser Ala Val Lys Ile Ala370 375 380 Glu Asp His Ser Asp Phe Val Ile Gly Phe Ile Ser Val Asn ProAla 385 390 395 400 Ser Trp Pro Gly Ala Pro Ile Asn Pro Ser Phe Ile GlnAla Thr Pro 405 410 415 Gly Val Gln Met Val Thr Gly Gly Asp Ala Leu GlyGln Gln Tyr Asn 420 425 430 Thr Pro Tyr Ser Val Ile His Asp Arg Gly SerAsp Ile Ile Ile Val 435 440 445 Gly Arg Gly Ile Ile Lys Ala Ala Asn ProAla Glu Ile Ala Arg Glu 450 455 460 Tyr Arg Leu Gln Gly Trp Asn Ala TyrLeu Ala Lys Cys Asn 465 470 475 7 1781 DNA Triticum aestivum 7gcacgagtct cgctccgccg ccgccgcctc cctccccagt cgatcaccaa acctcagtcc 60aaactccaaa cccccgccgc atcagaaaaa aaccctaggc catggacgcc gcggcgctgg 120agtcgctcat cctggacctc cacgccatcg aggtcgtgaa gctgggctcc ttcacgctca 180agtccggcat caaatcgccc atctacctcg acctccgcgc gctcgtctcc cacccgcgcc 240tgctctccgc cgtcgcctcg ctccttcacg cgctcccggc cacgcgcccc tacggcctcg 300tctgcggtgt cccctacacc gcgctcccca tcgccgccgt cctctccgtc gaccgctcaa 360tccccatgct catgcgccgc aaggaggtca aggcccacgg caccgccaag tccatcgagg 420gctccttcag ccccggggac accgtcctca tcatcgagga cctcgtcacc agtggcgcct 480ccgtgctcga gaccgccgcc ccgctccgcg ccgaggggct cgtcgtcgcc gacgccgtag 540tcgtcgtcga ccgcgagcag ggtggcaggg agaacctcgc cgctaatggg atcacgctgc 600actcgctcat gaccctcacg gaggtgctgg ccgtgctgct caagcacggg aaggtgaccg 660aggagaaggc gcaggaggtg aggcagttcc tcgacgccaa caggaaggtg gcggtgcctg 720gggcagcacc tgttacaccc agggtgctca gaaagacatt ttcggagagg gcgaatcttg 780ccaccaaccc tatggggaag aagctcttcg agctgatgga gaccaagcag accaacctgt 840gtgttgccgc tgatgtcggg acaacaaagg aactccttga gctggctgac aaggtcggcc 900ctcaaatttg tatgttgaaa acccatgtgg atatattatc tgattttacc ccagattttg 960gctctaagct ccgctcgatt gctgagaagc acaacttttt gatcttcgaa gaccgcaagt 1020ttgctgacat tggaaataca gtaaccatgc aatatgaagg aggaatattc cgcatattgg 1080attgggccga tattgttaat gcgcatatag tacctggacc tggaatcgta gatggcttga 1140agctgaaggg tttgcctaaa ggaagagggc tacttctgct cgctgagatg agctctgccg 1200gcaaccttgc ccatggagat tacactgctg ctgccgtaaa gattgctgag caacattctg 1260attttgtgat gggatttata tcagtaaatc ctgagtcttg gtcagtaaaa ccatcaagcc 1320ctgcatttat ccatgccacg cctggagttc agatggtcgc aggaggagat gatcttgggc 1380aacaatacaa cactcccgaa tctgtgataa actacagggg cagtgacata atcatagttg 1440gccgtgggat tataaaggcg agcgatccta tgaagaaggc gtgggagtac cgcttgcaag 1500ggtggcaggc atacaagaac agcttgctat gaaggaaggg gggcgccatg agcatcccca 1560agtataaggg cgaatccagt cagtttggcg aaataagcgc atgcggaaag gttttcctgc 1620agttgagtca ggacctaatt gacatcagat tcactgcaga ggagactcat gccccatcat 1680cgtttctgtt acaataattt cctctcggtt taccctgttc ttgctggttg agttaggcac 1740gttgtgatgc ctgtgcgcgg ttaaaaaaaa aaaaaaaaaa a 1781 8 476 PRT Triticumaestivum 8 Met Asp Ala Ala Ala Leu Glu Ser Leu Ile Leu Asp Leu His AlaIle 1 5 10 15 Glu Val Val Lys Leu Gly Ser Phe Thr Leu Lys Ser Gly IleLys Ser 20 25 30 Pro Ile Tyr Leu Asp Leu Arg Ala Leu Val Ser His Pro ArgLeu Leu 35 40 45 Ser Ala Val Ala Ser Leu Leu His Ala Leu Pro Ala Thr ArgPro Tyr 50 55 60 Gly Leu Val Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile AlaAla Val 65 70 75 80 Leu Ser Val Asp Arg Ser Ile Pro Met Leu Met Arg ArgLys Glu Val 85 90 95 Lys Ala His Gly Thr Ala Lys Ser Ile Glu Gly Ser PheSer Pro Gly 100 105 110 Asp Thr Val Leu Ile Ile Glu Asp Leu Val Thr SerGly Ala Ser Val 115 120 125 Leu Glu Thr Ala Ala Pro Leu Arg Ala Glu GlyLeu Val Val Ala Asp 130 135 140 Ala Val Val Val Val Asp Arg Glu Gln GlyGly Arg Glu Asn Leu Ala 145 150 155 160 Ala Asn Gly Ile Thr Leu His SerLeu Met Thr Leu Thr Glu Val Leu 165 170 175 Ala Val Leu Leu Lys His GlyLys Val Thr Glu Glu Lys Ala Gln Glu 180 185 190 Val Arg Gln Phe Leu AspAla Asn Arg Lys Val Ala Val Pro Gly Ala 195 200 205 Ala Pro Val Thr ProArg Val Leu Arg Lys Thr Phe Ser Glu Arg Ala 210 215 220 Asn Leu Ala ThrAsn Pro Met Gly Lys Lys Leu Phe Glu Leu Met Glu 225 230 235 240 Thr LysGln Thr Asn Leu Cys Val Ala Ala Asp Val Gly Thr Thr Lys 245 250 255 GluLeu Leu Glu Leu Ala Asp Lys Val Gly Pro Gln Ile Cys Met Leu 260 265 270Lys Thr His Val Asp Ile Leu Ser Asp Phe Thr Pro Asp Phe Gly Ser 275 280285 Lys Leu Arg Ser Ile Ala Glu Lys His Asn Phe Leu Ile Phe Glu Asp 290295 300 Arg Lys Phe Ala Asp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly305 310 315 320 Gly Ile Phe Arg Ile Leu Asp Trp Ala Asp Ile Val Asn AlaHis Ile 325 330 335 Val Pro Gly Pro Gly Ile Val Asp Gly Leu Lys Leu LysGly Leu Pro 340 345 350 Lys Gly Arg Gly Leu Leu Leu Leu Ala Glu Met SerSer Ala Gly Asn 355 360 365 Leu Ala His Gly Asp Tyr Thr Ala Ala Ala ValLys Ile Ala Glu Gln 370 375 380 His Ser Asp Phe Val Met Gly Phe Ile SerVal Asn Pro Glu Ser Trp 385 390 395 400 Ser Val Lys Pro Ser Ser Pro AlaPhe Ile His Ala Thr Pro Gly Val 405 410 415 Gln Met Val Ala Gly Gly AspAsp Leu Gly Gln Gln Tyr Asn Thr Pro 420 425 430 Glu Ser Val Ile Asn TyrArg Gly Ser Asp Ile Ile Ile Val Gly Arg 435 440 445 Gly Ile Ile Lys AlaSer Asp Pro Met Lys Lys Ala Trp Glu Tyr Arg 450 455 460 Leu Gln Gly TrpGln Ala Tyr Lys Asn Ser Leu Leu 465 470 475 9 1889 DNA Zea mays 9ccacgcgtcc gtacaaagcc acttcctgct ctcgctccgc cgccgccgcc tccctcccca 60gtcgatcacc aaacctcagt ccaaactcca aacccccgcc gcatcagaaa aaaaccctag 120gccatggacg ccgcggcgct ggagtcgctc atcctggacc tccacgccat cgaggtcgtg 180aagctgggct ccttcacgct caagtccggc atcaaatcgc ccatctacct cgacctccgc 240gcgctcgtct cccacccgcg cctgctctcc gccgtcgcct cgctccttca cgcgctcccg 300gccacgcgcc cctacggcct cgtctgcggt gtcccctaca ccgcgctccc catcgccgcc 360gtcctctccg tcgaccgctc aatccccatg ctcatgcgcc gcaaggaggt caaggcccac 420ggcaccgcca agtccatcga gggctccttc agccccgggg acaccgtcct catcatcgag 480gacctcgtca ccagtggcgc ctccgtgctc gagaccgccg ccccgctccg cgccgagggg 540ctcgtcgtcg ccgacgccgt agtcgtcgtc gaccgcgagc agggtggcag ggagaacctc 600gccgctaatg ggatcacgct gcactcgctc atgaccctca cggaggtgct ggccgtgctg 660ctcaagcacg ggaaggtgac cgaggagaag gcgcaggagg tgaggcagtt cctcgacgcc 720aacaggaagg tggcggtgcc tggggcagca cctgttacac ccagggtgct cagaaagaca 780ttttcggaga gggcgaatct tgccaccaac cctatgggga agaagctctt cgagctgatg 840gagaccaagc agaccaacct gtgtgttgcc gctgatgtcg ggacaacaaa ggaactcctt 900gagctggctg acaaggtcgg ccctcaaatt tgtatgttga aaacccatgt ggatatatta 960tctgatttta ccccagattt tggctctaag ctccgctcga ttgctgagaa gcacaacttt 1020ttgatcttcg aagaccgcaa gtttgctgac attggaaata cagtaaccat gcaatatgaa 1080ggaggaatat tccgcatatt ggattgggcc gatattgtta atgcgcatat agtacctgga 1140cctggaatcg tagatggctt gaagctgaag ggtttgccta aaggaagagg gctacttctg 1200ctcgctgaga tgagctctgc cggcaacctt gcccatggag attacactgc tgctgccgta 1260aagattgctg agcaacattc tgattttgtg atgggattta tatcagtaaa tcctgagtct 1320tggtcagtaa aaccatcaag ccctgcattt atccatgcca cgcctggagt tcagatggtc 1380gcaggaggag atgatcttgg gcaacaatac aacactcccg aatctgtgat aaactacagg 1440ggcagtgaca taatcatagt tggccgtggg attataaagg cgagcgatcc tatgaagaag 1500gcgtgggagt accgcttgca agggtggcag gcatacaaga acagcttgct atgaaggaag 1560gggggcgcca tgagcatccc caagtataag ggcgaatcca gtcagtttgg cgaaataagc 1620gcatgcggaa aggttttcct gcagttgagt caggacctaa ttgacatcag attcactgca 1680gaggagactc atgccccatc atcgtttctg ttacaataat ttcctctcgg tttaccctgt 1740tcttgctggt tgagttaggc acgttgtgat gcctgtgcgc ggttaaatcg tcttactgcc 1800atgccacttg aggtttggac tcttgagcaa gcaattttat cgatgccgag aattgtatga 1860aaaaaaaaaa aaaaaaaaaa aaaaaaaag 1889 10 476 PRT Zea mays 10 Met Asp AlaAla Ala Leu Glu Ser Leu Ile Leu Asp Leu His Ala Ile 1 5 10 15 Glu ValVal Lys Leu Gly Ser Phe Thr Leu Lys Ser Gly Ile Lys Ser 20 25 30 Pro IleTyr Leu Asp Leu Arg Ala Leu Val Ser His Pro Arg Leu Leu 35 40 45 Ser AlaVal Ala Ser Leu Leu His Ala Leu Pro Ala Thr Arg Pro Tyr 50 55 60 Gly LeuVal Cys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Ala Val 65 70 75 80 LeuSer Val Asp Arg Ser Ile Pro Met Leu Met Arg Arg Lys Glu Val 85 90 95 LysAla His Gly Thr Ala Lys Ser Ile Glu Gly Ser Phe Ser Pro Gly 100 105 110Asp Thr Val Leu Ile Ile Glu Asp Leu Val Thr Ser Gly Ala Ser Val 115 120125 Leu Glu Thr Ala Ala Pro Leu Arg Ala Glu Gly Leu Val Val Ala Asp 130135 140 Ala Val Val Val Val Asp Arg Glu Gln Gly Gly Arg Glu Asn Leu Ala145 150 155 160 Ala Asn Gly Ile Thr Leu His Ser Leu Met Thr Leu Thr GluVal Leu 165 170 175 Ala Val Leu Leu Lys His Gly Lys Val Thr Glu Glu LysAla Gln Glu 180 185 190 Val Arg Gln Phe Leu Asp Ala Asn Arg Lys Val AlaVal Pro Gly Ala 195 200 205 Ala Pro Val Thr Pro Arg Val Leu Arg Lys ThrPhe Ser Glu Arg Ala 210 215 220 Asn Leu Ala Thr Asn Pro Met Gly Lys LysLeu Phe Glu Leu Met Glu 225 230 235 240 Thr Lys Gln Thr Asn Leu Cys ValAla Ala Asp Val Gly Thr Thr Lys 245 250 255 Glu Leu Leu Glu Leu Ala AspLys Val Gly Pro Gln Ile Cys Met Leu 260 265 270 Lys Thr His Val Asp IleLeu Ser Asp Phe Thr Pro Asp Phe Gly Ser 275 280 285 Lys Leu Arg Ser IleAla Glu Lys His Asn Phe Leu Ile Phe Glu Asp 290 295 300 Arg Lys Phe AlaAsp Ile Gly Asn Thr Val Thr Met Gln Tyr Glu Gly 305 310 315 320 Gly IlePhe Arg Ile Leu Asp Trp Ala Asp Ile Val Asn Ala His Ile 325 330 335 ValPro Gly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly Leu Pro 340 345 350Lys Gly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser Ser Ala Gly Asn 355 360365 Leu Ala His Gly Asp Tyr Thr Ala Ala Ala Val Lys Ile Ala Glu Gln 370375 380 His Ser Asp Phe Val Met Gly Phe Ile Ser Val Asn Pro Glu Ser Trp385 390 395 400 Ser Val Lys Pro Ser Ser Pro Ala Phe Ile His Ala Thr ProGly Val 405 410 415 Gln Met Val Ala Gly Gly Asp Asp Leu Gly Gln Gln TyrAsn Thr Pro 420 425 430 Glu Ser Val Ile Asn Tyr Arg Gly Ser Asp Ile IleIle Val Gly Arg 435 440 445 Gly Ile Ile Lys Ala Ser Asp Pro Met Lys LysAla Trp Glu Tyr Arg 450 455 460 Leu Gln Gly Trp Gln Ala Tyr Lys Asn SerLeu Leu 465 470 475 11 1627 DNA Oryza sativa unsure (1489) n = A, C, Gor T 11 gcacgagctt acacccgccc aaaaccctag ctaagcctag ccgccatggacgccgccgcg 60 caggaatccc tcatcctgga gctccacgcc atcgaggcca tcaagttcggcaccttcgtg 120 ctcaagtccg gcatcacctc cccgatctac ctcgacctcc gcgcgctcgtctcccacccg 180 ggcctcctct cctccatcgc caccctcctc cacaccctcc cggcgacccgcccctacgac 240 ctcctctgcg gcgtccccta caccgcgctc cccatcgcct ccgtcctctccgtccaccgc 300 tccgtcccca tggtcatgcg ccgcaaggag gccaaggccc acggcaccgccaagtccatc 360 gagggcgcct tccgcgccgg ggaggccgtg ctcatcatcg aggacctcgtcaccagcggc 420 gcctccgttc tcgagaccgc cgcgccgctc cgcgaccagg ggctcgtcgtcgccgacgcc 480 gtcgtcgtcg tcgaccgcaa gcagggcggg agggagaacc ttgccgccaatgggatcacg 540 ctgcactcgc tcatgaccct cacggaggtg ctcgccgtgc tgctcaagcacgggaaggtg 600 acccaagaag agcgaggagg taagcagttt cttgacgcca ataggaaggtgaccgttccc 660 ggagcggcgg gcgccgttaa gcccaaagcg gtcaggaagg ggtttgctgagagggctgga 720 ttggccaaga acccgatggg gaagaggctt ttcgaggtga tggaggcaaagcagagcaat 780 ttatgtgttg ctgccgatgt gggaactgca aaggagctcc ttgagcttgcagagaaggtt 840 ggtccagaga tttgcatgct gaaaactcat gtggatatct tgtctgactttactccagat 900 tttggagcta agcttcgctc gattgccgag aagcacaact ttttgatatttgaagaccgc 960 aagtttgctg acattggaaa cacagtgact atgcaatatg aaggaggaatatttcgcata 1020 ttagactggg ctgatatcgt caatgcccat ataattcctg gacctggaattgtggatggt 1080 ctgaagctta agggtttgcc aaaaggaaga gggctgcttt tgcttgctgaaatgagctcg 1140 gctggcaacc ttgctcatgg agagtacact gctgcagctg taaagattgctgagcaacat 1200 tctgattttg taattggatt tatatccgtt aatccagcat cttggtcagttgcgccatca 1260 agtccagcat ttatccatgc cactcctgga gtgcagatgg tttctggaggagatgctctt 1320 ggtcaacagt acaatacccc tcattctgtt ataaacgaca agaggcaagtgacataatta 1380 tagtccggac gagggattat aaaggcgaag taatccagcc cgagaccgcgagggaagtac 1440 cgcatccaag ggtgggggag caaaacaatc cagctttgcc atgagaaantgagaatngtg 1500 tttaggcaat ggttggttcn agcttatgat ttattataac caagaataattaagccanga 1560 ttgcnnataa agccgggatt aatantnaag ctgccatana aataaactgtgnagttggtt 1620 gntttgg 1627 12 443 PRT Oryza sativa 12 Met Asp Ala AlaAla Gln Glu Ser Leu Ile Leu Glu Leu His Ala Ile 1 5 10 15 Glu Ala IleLys Phe Gly Thr Phe Val Leu Lys Ser Gly Ile Thr Ser 20 25 30 Pro Ile TyrLeu Asp Leu Arg Ala Leu Val Ser His Pro Gly Leu Leu 35 40 45 Ser Ser IleAla Thr Leu Leu His Thr Leu Pro Ala Thr Arg Pro Tyr 50 55 60 Asp Leu LeuCys Gly Val Pro Tyr Thr Ala Leu Pro Ile Ala Ser Val 65 70 75 80 Leu SerVal His Arg Ser Val Pro Met Val Met Arg Arg Lys Glu Ala 85 90 95 Lys AlaHis Gly Thr Ala Lys Ser Ile Glu Gly Ala Phe Arg Ala Gly 100 105 110 GluAla Val Leu Ile Ile Glu Asp Leu Val Thr Ser Gly Ala Ser Val 115 120 125Leu Glu Thr Ala Ala Pro Leu Arg Asp Gln Gly Leu Val Val Ala Asp 130 135140 Ala Val Val Val Val Asp Arg Lys Gln Gly Gly Arg Glu Asn Leu Ala 145150 155 160 Ala Asn Gly Ile Thr Leu His Ser Leu Met Thr Leu Thr Glu ValLeu 165 170 175 Ala Val Leu Leu Lys His Gly Lys Val Thr Gln Glu Glu ArgGly Gly 180 185 190 Lys Gln Phe Leu Asp Ala Asn Arg Lys Val Thr Val ProGly Ala Ala 195 200 205 Gly Ala Val Lys Pro Lys Ala Val Arg Lys Gly PheAla Glu Arg Ala 210 215 220 Gly Leu Ala Lys Asn Pro Met Gly Lys Arg LeuPhe Glu Val Met Glu 225 230 235 240 Ala Lys Gln Ser Asn Leu Cys Val AlaAla Asp Val Gly Thr Ala Lys 245 250 255 Glu Leu Leu Glu Leu Ala Glu LysVal Gly Pro Glu Ile Cys Met Leu 260 265 270 Lys Thr His Val Asp Ile LeuSer Asp Phe Thr Pro Asp Phe Gly Ala 275 280 285 Lys Leu Arg Ser Ile AlaGlu Lys His Asn Phe Leu Ile Phe Glu Asp 290 295 300 Arg Lys Phe Ala AspIle Gly Asn Thr Val Thr Met Gln Tyr Glu Gly 305 310 315 320 Gly Ile PheArg Ile Leu Asp Trp Ala Asp Ile Val Asn Ala His Ile 325 330 335 Ile ProGly Pro Gly Ile Val Asp Gly Leu Lys Leu Lys Gly Leu Pro 340 345 350 LysGly Arg Gly Leu Leu Leu Leu Ala Glu Met Ser Ser Ala Gly Asn 355 360 365Leu Ala His Gly Glu Tyr Thr Ala Ala Ala Val Lys Ile Ala Glu Gln 370 375380 His Ser Asp Phe Val Ile Gly Phe Ile Ser Val Asn Pro Ala Ser Trp 385390 395 400 Ser Val Ala Pro Ser Ser Pro Ala Phe Ile His Ala Thr Pro GlyVal 405 410 415 Gln Met Val Ser Gly Gly Asp Ala Leu Gly Gln Gln Tyr AsnThr Pro 420 425 430 His Ser Val Ile Asn Asp Lys Arg Gln Val Thr 435 44013 476 PRT Arabidopsis thaliana 13 Met Ser Ala Met Glu Ala Leu Ile LeuGln Leu His Glu Ile Gly Ala 1 5 10 15 Val Lys Phe Gly Asn Phe Lys LeuLys Ser Gly Ile Phe Ser Pro Val 20 25 30 Tyr Ile Asp Leu Arg Leu Ile ValSer Tyr Pro Ser Leu Leu Thr Gln 35 40 45 Ile Ser Gln Thr Leu Ile Ser SerLeu Pro Pro Ser Ala Thr Phe Asp 50 55 60 Val Val Cys Gly Val Pro Tyr ThrAla Leu Pro Ile Ala Thr Val Val 65 70 75 80 Ser Val Ser Asn Gly Ile ProMet Leu Met Arg Arg Lys Glu Ile Lys 85 90 95 Asp Tyr Gly Thr Ser Lys AlaIle Glu Gly Ile Phe Glu Lys Asp Gln 100 105 110 Thr Cys Leu Ile Ile GluAsp Leu Val Thr Ser Gly Ala Ser Val Leu 115 120 125 Glu Thr Ala Ala ProLeu Arg Ala Val Gly Leu Lys Val Ser Asp Ala 130 135 140 Val Val Leu IleAsp Arg Gln Gln Gly Gly Arg Glu Asn Leu Ala Glu 145 150 155 160 Asn GlyIle Lys Leu His Ser Met Ile Met Leu Thr Asp Met Val Arg 165 170 175 ValLeu Lys Glu Lys Gly Lys Ile Glu Glu Glu Val Glu Val Asn Leu 180 185 190Leu Lys Phe Leu Glu Glu Asn Arg Arg Val Ser Val Pro Ser Val Glu 195 200205 Lys Pro Lys Pro Lys Pro Arg Val Leu Gly Phe Lys Glu Arg Ser Glu 210215 220 Leu Ser Lys Asn Pro Thr Gly Lys Lys Leu Phe Asp Ile Met Leu Lys225 230 235 240 Lys Glu Thr Asn Leu Cys Leu Ala Ala Asp Val Gly Thr AlaAla Glu 245 250 255 Leu Leu Asp Ile Ala Asp Lys Val Gly Pro Glu Ile CysLeu Leu Lys 260 265 270 Thr His Val Asp Ile Leu Pro Asp Phe Thr Pro AspPhe Gly Ser Lys 275 280 285 Leu Arg Ala Ile Ala Asp Lys His Lys Phe LeuIle Phe Glu Asp Arg 290 295 300 Lys Phe Ala Asp Ile Gly Asn Thr Val ThrMet Gln Tyr Glu Gly Gly 305 310 315 320 Ile Phe Lys Ile Leu Glu Trp AlaAsp Ile Ile Asn Ala His Val Ile 325 330 335 Ser Gly Pro Gly Ile Val AspGly Leu Lys Leu Lys Gly Met Pro Arg 340 345 350 Gly Arg Gly Leu Leu LeuLeu Ala Glu Met Ser Ser Ala Gly Asn Leu 355 360 365 Ala Thr Gly Asp TyrThr Ala Ala Ala Val Lys Ile Ala Asp Ala His 370 375 380 Ser Asp Phe ValMet Gly Phe Ile Ser Val Asn Pro Ala Ser Trp Lys 385 390 395 400 Cys GlyTyr Val Tyr Pro Ser Met Ile His Ala Thr Pro Gly Val Gln 405 410 415 MetVal Lys Gly Gly Asp Ala Leu Gly Gln Gln Tyr Asn Thr Pro His 420 425 430Ser Val Ile Thr Glu Arg Gly Ser Asp Ile Ile Ile Val Gly Arg Gly 435 440445 Ile Ile Lys Ala Glu Asn Pro Ala Glu Thr Ala His Glu Tyr Arg Val 450455 460 Gln Gly Trp Asn Ala Tyr Leu Glu Lys Cys Ser Gln 465 470 475 14476 PRT Nicotiana tabacum 14 Met Ser Ala Met Glu Ala Leu Ile Leu Gln LeuHis Glu Ile Gly Ala 1 5 10 15 Val Lys Phe Gly Asn Phe Lys Leu Lys SerGly Ile Phe Ser Pro Val 20 25 30 Tyr Ile Asp Leu Arg Leu Ile Val Ser TyrPro Ser Leu Leu Thr Gln 35 40 45 Ile Ser Gln Thr Leu Ile Ser Ser Leu ProPro Ser Ala Thr Phe Asp 50 55 60 Val Val Cys Gly Val Pro Tyr Thr Ala LeuPro Ile Ala Thr Val Val 65 70 75 80 Ser Val Ser Asn Gly Ile Pro Met LeuMet Arg Arg Lys Glu Ile Lys 85 90 95 Asp Tyr Gly Thr Ser Lys Ala Ile GluGly Ile Phe Glu Lys Asp Gln 100 105 110 Thr Cys Leu Ile Ile Glu Asp LeuVal Thr Ser Gly Ala Ser Val Leu 115 120 125 Glu Thr Ala Ala Pro Leu ArgAla Val Gly Leu Lys Val Ser Asp Ala 130 135 140 Val Val Leu Ile Asp ArgGln Gln Gly Gly Arg Glu Asn Leu Ala Glu 145 150 155 160 Asn Gly Ile LysLeu His Ser Met Ile Met Leu Thr Asp Met Val Arg 165 170 175 Val Leu LysGlu Lys Gly Lys Ile Glu Glu Glu Val Glu Val Asn Leu 180 185 190 Leu LysPhe Leu Glu Glu Asn Arg Arg Val Ser Val Pro Ser Val Glu 195 200 205 LysPro Lys Pro Lys Pro Arg Val Leu Gly Phe Lys Glu Arg Ser Glu 210 215 220Leu Ser Lys Asn Pro Thr Gly Lys Lys Leu Phe Asp Ile Met Leu Lys 225 230235 240 Lys Glu Thr Asn Leu Cys Leu Ala Ala Asp Val Gly Thr Ala Ala Glu245 250 255 Leu Leu Asp Ile Ala Asp Lys Val Gly Pro Glu Ile Cys Leu LeuLys 260 265 270 Thr His Val Asp Ile Leu Pro Asp Phe Thr Pro Asp Phe GlySer Lys 275 280 285 Leu Arg Ala Ile Ala Asp Lys His Lys Phe Leu Ile PheGlu Asp Arg 290 295 300 Lys Phe Ala Asp Ile Gly Asn Thr Val Thr Met GlnTyr Glu Gly Gly 305 310 315 320 Ile Phe Lys Ile Leu Glu Trp Ala Asp IleIle Asn Ala His Val Ile 325 330 335 Ser Gly Pro Gly Ile Val Asp Gly LeuLys Leu Lys Gly Met Pro Arg 340 345 350 Gly Arg Gly Leu Leu Leu Leu AlaGlu Met Ser Ser Ala Gly Asn Leu 355 360 365 Ala Thr Gly Asp Tyr Thr AlaAla Ala Val Lys Ile Ala Asp Ala His 370 375 380 Ser Asp Phe Val Met GlyPhe Ile Ser Val Asn Pro Ala Ser Trp Lys 385 390 395 400 Cys Gly Tyr ValTyr Pro Ser Met Ile His Ala Thr Pro Gly Val Gln 405 410 415 Met Val LysGly Gly Asp Ala Leu Gly Gln Gln Tyr Asn Thr Pro His 420 425 430 Ser ValIle Thr Glu Arg Gly Ser Asp Ile Ile Ile Val Gly Arg Gly 435 440 445 IleIle Lys Ala Glu Asn Pro Ala Glu Thr Ala His Glu Tyr Arg Val 450 455 460Gln Gly Trp Asn Ala Tyr Leu Glu Lys Cys Ser Gln 465 470 475

What is claimed is:
 1. An isolated polynucleotide that encodes apolypeptide of at least 200 amino acids having a sequence identity of atleast 85% based on the Clustal method of alignment when compared to apolypeptide selected from the group consisting of SEQ ID NOs: 6, 8, 10,and
 12. 2. A polynucleotide sequence of claim 1, wherein sequenceidentity is at least 90%.
 3. A polynucleotide sequence of claim 1,wherein sequence identity is at least 95%.
 4. The polynucleotide ofclaim 1 wherein the polynucleotide encodes a polypeptide selected fromthe group consisting of SEQ ID NOs: 6, 8, 10 and
 12. 5. Thepolynucleotide of claim 1, wherein the polynucleotide comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 5,7, 9 and
 11. 6. The polynucleotide of claim 1, wherein the polypeptideis an OMP decarboxylase.
 7. An isolated complement of the polynucleotideof claim 1, wherein (a) the complement and the polynucleotide consist ofthe same number of nucleotides, and (b) the nucleotide sequences of thecomplement and the polynucleotide have 100% complementanty.
 8. Anisolated nucleic acid molecule that (1) comprises at least 800nucleotides and (2) remains hybridized with the isolated polynucleotideof claim 24 under a washing condition of 0.1×SSC, 0.1% SDS, and 650 C.9. A cell comprising the polynucleotide of claim
 1. 10. The cell ofclaim 9, wherein the cell is selected from the group consisting of ayeast cell, a bacterial cell and a plant cell.
 11. A transgenic plantcomprising the polynucleotide of claim
 1. 12. A method for transforminga cell comprising introducing into a cell the polynucleotide of claim 1.13. A method for producing a transgenic plant comprising (a)transforming a plant cell with the polynucleotide of claim 1, and (b)regenerating a plant from the transformed plant cell.
 14. A method forproducing a polynucleotide fragment comprising (a) selecting anucleotide sequence comprised by the polynucleotide of claim 1, and (b)synthesizing a polynucleotide fragment containing the nucleotidesequence.
 15. The method of claim 14, wherein the fragment is producedin vivo.
 16. An isolated polypeptide comprising (a) at least 200 aminoacids, and (b) a first amino acid sequence, wherein the first amino acidsequence and a second amino acid sequence have a sequence identity of atleast 85%, and wherein the second amino acid is selected from the groupconsisting of SEQ ID NOs: 6, 8, 10, and
 12. 17. The polypeptide of claim16, wherein the sequence identity is at least 90%.
 18. The polypeptideof claim 16, wherein the sequence identity is at least 95%.
 19. Thepolypeptide of claim 16 wherein the polypeptide has a sequence selectedfrom the group consisting of SEQ ID NOs: 6, 8, 10, and
 12. 20. Thepolypeptide of claim 16, wherein the polypeptide is an OMPdecarboxylase.
 21. A chimeric gene comprising the polynucleotide ofclaim 1 operably linked to at least one suitable regulatory sequence.22. A method for altering the level of OMP decarboxylase expression in ahost cell, the method comprising: (a) Transforming a host cell with thechimeric gene of claim 21; and (b) Growing the transformed cell in step(a) under conditions suitable for the expression of the chimeric gene.